Therapeutic drugs (thereafter also referred to as “drug” or “drugs”) can be either natural products, or small molecule drugs, or peptides, or therapeutic proteins (biotherapeutics), or small-molecule-biotherapeutic conjugates (Barbosa, M. D. F. S. et al. 2013 Anal. Biochem. 441: 174-179; Barbosa, M. D. F. S. and Smith, D. D. 2014 Drug Discov. Today 19: 1897-1912; Woodcock, J. et al. 2007 Nat. Rev. Drug Discov. 6: 437-442; all expressly incorporated by reference herein). Combination therapies (in which more than one entity is used) are also common.
In attempts to improve efficacy and/or to protect intellectual property positions, several new versions of marketed therapeutic drugs have been developed. In some instances, the novelty consists of introducing mutations to existing protein drugs. For example, new insulins are now available for treatment of diabetes, which contain mutated protein sequences relative to native insulin. Protein mutations may significantly alter the drug properties (including but not restricted to aggregation propensity), and may also create epitopes involved in T cell activation and unwanted anti-drug antibody (ADA) responses. Unwanted immunogenicity is also a concern for biosimilar versions of marketed protein drugs, typically requiring postmarketing surveillance (a biosimilar is a biotherapeutic similar to another one already marketed for which the patent has expired).
Despite attempts to standardize ADA detection across the pharmaceutical industry and to determine its impact, a holistic method for doing so is lacking. Unifying approaches to solve that challenge, as described in some embodiments of the present invention, have been non-obvious. That non-obviousness is exemplified by what has been for more than a decade the common practice of having language in the labels of biotherapeutics approved by Regulatory Agencies (such as the U.S. Food and Drug Administration—FDA), to the effect that it would be misleading to compare immunogenicity data amongst products, due to factors such as differences in assays, and lack of standardization of sample handling and collection (Barbosa, M. D. F. S. and Smith, D. D. 2014 Drug Discov. Today 19: 1897-1912; Expressly incorporated by reference herein). Those challenges are becoming increasingly complex, as a growing number of products are approved by regulatory agencies, including but not restricted to biosimilars and modified versions of marketed biotherapeutics. Although monitoring ADAs is typically a regulatory requirement for development and approval of protein drugs, it has been difficult to standardize and unify testing procedures for the drugs approved, including related biotherapeutics (different insulin versions are non-limiting examples). During the development of a biosimilar, the same assay may be used to test ADA for comparison of the biosimilar with the reference product, but typically there has been no systematic postmarketing comparison with the reference product. Also importantly, in other situations there have been no mechanisms in place to compare systematically therapeutic drugs approved for the same application regarding their immunogenicity in humans, and one of the difficulties is that the assays used may vary from one product to the other, and may be validated differently in various laboratories (Barbosa, M. D. F. S. and Smith, D. D. 2014 Drug Discov. Today 19: 1897-1912; expressly incorporated by reference herein).
Assays that detect known analytes have been widely used during pharmacokinetic studies (hereinafter “PK” assay). PK assays have been more amenable to standardization, as the positive control is representative of the analyte in the sample tested, allowing for quantification. On the contrary, ADA responses are typically polyclonal, and each sample contains an unknown ADA repertoire. Hence, there is no positive control that accurately reflects the analytes in the samples, and those ADA assays are qualitative. This further complicates comparison of immunogenicity of different biotherapeutics.
The present invention describes a novel portable device that addresses the need for standardization of ADA testing across various therapeutic drugs, and immunogenicity comparisons. Although Plavina et at. (U.S. Pat. No. 9,377,458; expressly incorporated by reference herein) describes a two-step bridging assay for the detection of anti-anti-VL4 antibodies, the labeled detection reagent in that assay is the drug itself. What follows is that the assay format used to test anti-anti-VL4 antibodies would require independent batches of labeled reagents for testing of different drugs, thus introducing concerns similar to the ones for existing assays, regarding comparing immunogenicity of biotherapeutics.
ADAs may negatively impact efficacy and/or safety of the drug (Barbosa, M. D. F. S. and Smith, D. D. 2014 Drug Discov. Today 19: 1897-1912; expressly incorporated by reference herein). Antibody responses against adalimumab and infliximab are non-limiting examples of ADAs that have been shown to decrease or abolish the efficacy of those therapeutic drugs. Adalimumab and infliximab are IgG1 antibodies that differ regarding their variable region, although they both are tumor necrosis factor (TFN) blockers. It is typically not know if antibodies against the variable region of one of those two therapeutic drugs would cross-react with the other, or which one of those drugs is more immunogenic. Language in the labels of those therapeutic drugs (as approved by regulatory agencies) suggest that comparisons of the immunogenicity of either product would not be accurate, due to factors such as assay differences and sample handling (Barbosa, M. D. F. S. and Smith, D. D. 2014 Drug Discov. Today 19: 1897-1912; expressly incorporated by reference herein).
Hosts such as humans and test animals can also mount ADA responses against entities added to therapeutic proteins. For example, anti-polyethylene glycol (anti-PEG) ADAs have been often observed when hosts are dosed with therapeutic drug-PEG conjugates (Barbosa, M. D. F. S. et al. 2013 Anal. Biochem. 441: 174-179; expressly incorporated by reference herein). Furthermore, the ADAs may be specific for drug degradation products.
A competent host immune system may mount unwanted responses to therapeutic drugs, such as the formation of neutralizing and/or non-neutralizing ADAs and/or various types of hypersensitivity (Barbosa, M. D. F. S. and Smith, D. D. 2014 Drug Discov. Today. 19: 1897-1912; expressly incorporated by reference herein). Host immune reactions often play an important role in adverse effects of therapeutic drugs. Various adverse reactions can result from the use of therapeutic drugs, for example life-threatening IgE- or IgG-mediated anaphylaxis or anaphylactic shock (Barbosa, M. D. F. S. and Smith, D. D. 2014 Drug Discov. Today. 19: 1897-1912; expressly incorporated by reference herein). Although immunogenicity may be associated with all drug classes, the main focus has been in immunogenicity of biologic drugs, likely due to their documented magnitude compared to immunogenicity of small molecules (Barbosa, M. D. F. S. and Smith, D. D. 2014 Drug Discov. Today. 19: 1897-1912; expressly incorporated by reference herein). ADAs may cause clinical syndromes ranging from mild hypersensitivity reactions to life-threatening responses, and may also decrease efficacy of the drug by directly neutralizing activity or by increasing drug clearance (Barbosa, M. D. F. S. and Smith, D. D. 2014 Drug Discov. Today. 19: 1897-1912; expressly incorporated by reference herein).
Antibodies (also named immunoglobulins) are proteins that bind a specific antigen. In mammals such as humans and mice, antibodies contain paired heavy and light polypeptide chains. Standard antibody structural units typically comprise a tetramer. Each tetramer is usually composed of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of approximately 50 kDa). “Isotype” as used herein is meant any of the subclasses of immunoglobulins. The known human antibody isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.
Each antibody chain contains a variable and a constant region, as described above. The variable regions of the light and heavy chains are required for binding the target molecule (the antigen). All ADAs are capable of binding to a target molecule, and hence are referred to as binding antibodies.
An ADA that blocks or diminishes activity of the target protein is designated as a neutralizing antibody, commonly abbreviated to NAb. While some IgM can be neutralizing, usually most neutralizing ADAs (NAbs) are of the IgG type.
The following Igs are typically observed in higher mammals: IgD, IgA, IgE, IgM and IgG. IgD amounts to a small percentage of total serum Igs (less than 1%); IgA and IgM can comprise approximately 10-20%. IgG is the predominant Ig in blood. IgM is generally known as the early antibody, as it precedes the IgG response.
Host antibody responses against an antigen are typically polyclonal, comprising immunoglobulins that bind the antigen with various affinities and/or avidities. Hence, the assays used to detect antibody responses against therapeutic drugs are inherently qualitative, because there is no positive control antibody that would accurately represent all diverse antibodies in each of the samples collected from diverse sources and/or at various times following antigen exposure.
Another difficulty associated with monitoring ADAs for approved products is the cumbersome nature of collecting patient blood and shipping samples (commonly plasma or serum after blood processing) under special conditions to labs approved for such testing, and the lack of unified methodologies at such laboratories. In addition, such procedures are expensive and time-consuming, and in many instances laboratories offering those services are not even available and/or not known to physicians and/or patients. What follows is that there is an unmet need for devices to readily detect ADAs and to perform risk assessment for biotherapeutics. Such devices can have several utilities, including but not restricted to stratification of patients likely to benefit from a given therapy, comparison of similar products marketed for the same indication, guidance for new product development, tests during clinical trials, and postmarketing surveillance.
The difficulties associated with implementation of current approaches to postmarketing assessment of therapeutic drugs has been reviewed (Barbosa, M. D. F. S. and Smith, D. D. 2014 Drug Discov. Today 19: 1897-1912; expressly incorporated by reference herein). The US Food and Drug Administration recently initiated an active surveillance system (“the Sentinel Initiative”). However, one of the challenges associated with some aspects of drug comparisons with that system is that, in many cases, various different assays are used, resulting in data that is not amenable to the computational analysis. Such is the case for ADA assays.
With a plethora of therapeutic drugs being approved for the same indication, it is becoming increasingly complex for physicians and patients to select the medication likely to provide most benefits. For instance, several formulations of interferon-β (IFN-β) are marketed (Rebif®, Betaseron®/Betaferon®, Avonex®, and Pelegridy®), and recently IFN-β biosimilars are also being approved (Barbosa, M. D. F. S. and Smith, D. D. 2014 Drug Discov. Today 19: 1897-1912; expressly incorporated by reference herein). Devices to detect ADAs, as described in the present invention, would allow effective comparison of similar products marketed for the same indication, and/or to comparison of different products regarding suitability for specific patients.
ADA incidence against chronically administered products such as insulin and enzyme replacement therapies is also a concern. Even if the drug dosage is increased to compensate for NAbs, the chronic administration may result in immune complexes not being cleared, leading to immune complex disease and/or other syndromes (Barbosa, M. D. F. S. and Smith, D. D. 2014 Drug Discov. Today 19: 1897-1912; expressly incorporated by reference herein). In such cases, knowledge of ADA incidence and monitoring can provide an effective mechanism to evaluate risk and the need for tolerance induction regimens. Methods to assess risk of immune responses can also be useful to guide therapies other than the ones requiring chronic administration.
The present invention provides portable devices for evaluation of ADA responses against therapeutic drugs. Those devices can be used for one or more of the following: to stratify patients prior to therapy; to monitor efficacy of therapy; to monitor therapy safety; to guide discovery of novel therapeutic drugs; to guide therapeutic drug development; to estimate possibility of adverse events; to compare therapeutic drugs; to estimate need for tolerance induction; to empower doctors and patients regarding treatment decisions; for postmarketing surveillance.